Scholars Research Library. Study of the effect of Ampicillin Trihydrate on protein binding of Oseltamivir Phosphate

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1 Available online at Der Pharmacia Lettre, 2011, 3(3): ( ISSN USA CODEN: DPLEB4 Study of the effect of Ampicillin Trihydrate on protein binding of Oseltamivir Phosphate S. Vijayaraj*, Divya. S, K. Guru Prasad Varma, V. Pavana Keerthi, R. Pragna and M. Venkatesh Department of Pharmaceutical Analysis, Sree Vidyanikethan College of Pharmacy, Sree Sainath Nagar, Tirupathi ABSTRACT Protein bound drugs are pharmacokinetically inactive where unbound drugs are pharmacologically active.highly protein bound drugs have reduced Pharmacological effect (invitro) with Albumin. Free drug concentration in the plasma is responsible for the observed pharmacological effect or therapeutic response. Oseltamivir phosphate is an antiviral drug used in the treatment and prophylaxis of both influenza virus A and influenza virus B. It is currently the drug of choice for the prevention of influenza virus infection. Invitro Protein binding study of oseltamivir phosphate was carried out for 200 mins and The percentage of bound drug was found to be 80% and percentage of free drug was found to be 20%.In co-administration of Oseltamivir phosphate with Ampicillin, The percentage of bound drug was found to be 68.6% and percentage of free drug was found to be 31.40% Increase in percentage of free drug was found to be 57%, shows beneficial drug interaction. Keywords: Oseltamivir phosphate, Ampicillin trihydrate, protein binding, Co-administration INTRODUCTION Oseltamivir phosphate is an antiviral drug used in the treatment and prophylaxis of both influenza virus A and influenza virus B. Fig 1 Fig 1 : Structure of Oseltamivir phosphate 320

2 Oseltamivir phosphate is readily absorbed and rapidly converted (half-life [t 1/2 ], 1 to 3 h) to its active carboxylate metabolite via hepatic esterases [1]. At least 75% of an oral dose reaches the systemic circulation as oseltamivir carboxylate, while less than 5% of an oral dose reaches the systemic circulation as oseltamivir phosphate. Once it is formed, oseltamivir carboxylate is minimally bound (3%) to human plasma proteins. It has a t 1/2 of 6 to 10 h and is eliminated by the kidney by a first-order process that includes glomerular filtration and tubular secretion by an anionic transporter system. [2] Ampicillin is a beta-lactam antibiotic that has been used extensively to treat bacterial infections. [3] Fig 2. Fig 2: Structure of Ampicillin trihydrate Co-administration of Oseltamivir phosphate with Ampicillin trihydrate shows beneficial drug interaction, thus increase bioavailability of oseltamivir phosphate. MATERIALS AND METHODS Equipment Shimadzu UV Pharma Spectrophotometer 1700, Elico L1613 ph Meter Shimadzu (ELB 300) Electronic balance, Shimadzu (BL 22OH) Electronic balance Reagents and solutions All employed chemicals were of analytical grade and high-purified water was used throughout. Oseltamivir phosphate pure sample was obtained as a gift sample from Hetero drugs, Hyderabad, India. The gift sample of pure drug Ampicillin trihydrate was received from Parabolic drugs. Ltd. Preparation of the standard graph of Oseltamivir Phosphate: 1) Preparation of primary stock solution: 10mg of oseltamivir phosphate taken in 100ml standard flask and make up the volume with 7.2 ph Phosphate buffer.this gives 100µg/ml.This is the primary stock solution. 2) Preparation of various concentrations of oseltamivir phosphate: From the primary stock solution the concentrations ranging from 15 µg/ml to 75µg/ml was prepared by diluting with phosphate buffer. A Standard graph was plotted by taking Time on X- axis and Absorbance on Y-axis. Fig 3 321

3 Fig-3: Standard graph of Oseltamivir phosphate Calibration curve for protein binding Absorbance Concentration(mcg/ml) Preparation of 2.8 X 10-4 M Solution of Egg Albumin g of egg albumin flakes is dissolved in 25 ml of distilled water. It is shaken well and is kept aside. Table-1: Protein binding studies of oseltamivir phosphate and ampicillin trihydrate Time(mins) Absorbance of M Oseltamivir phosphate Absorbance of the mixture of Oseltamivir phosphate and Ampicillin Studies on protein binding: 25 ml OF 1.63 X 10-4 M of oseltamivir phosphate is prepared using a buffer of ph 7.2. A boiling tube open on both sides is taken and a semipermeable membrane is tied on to the neck of the boiling tube. The egg albumin solution 10ml is taken inside the semipermeable membrane. The boiling tube is then immersed into the beaker containing the drug Oseltamivir phosphate 1.63 X 10-4 M. Immediately at zero time, 1ml of solution is pipetted out from the beaker and is replaced with 1ml of water. Readings are taken at 10, 20, 40, 60, 80, 100, 120 min and absorbance at 525 nm and is noted. Once equilibrium is reached there will be no further change in absorbance.so the constant value of absorbance is noted. From the absorbance, by using the standard graph, 322

4 determine the concentration in mcg/ml by extrapolation. From the concentration in mcg/ml determine the concentration in moles. [4] Table1 Preparation of M solution of oseltamivir phosphate: gms of drug dissolved in 1000ml of distilled water to obtain 1M.For M solution 0.067gms of drug dissolved in 1000ml of distilled water. Procedure From the prepared oseltamivir phosphate solution 15ml solution was taken in a beaker and 15ml of albumin solution in boiling test separated by membrane.1ml of drug solution was taken from beaker for every 20mins and replaced with buffer immediately.1ml of solution was diluted to 10ml of buffer and absorbance was noted until a constant value by using UV spectrophotometer. Preparation of M solution of Ampicillin trihydrate: Gms of drug dissolved in 1000ml of distilled water to obtain 1M. For M solution gms of drug dissolved in 1000ml of distilled water. Table 2: Percentage of free drug of oseltamivir phosphate in presence of ampicillin trihydrate Drug oseltamivir phosphate oseltamivir phosphate and ampicillin % drug bound 80% 68.6% %drug free 20% 31.4% % increase in free drug 11.4% Fig 4 protein binding study of oseltamivir phosphate %DRUG 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% 80% 20% oseltamivir phosphate 68.60% 31.40% oseltamivir phosphate and ampicillin CONDITION OF PROTEIN BINDING % drug bound %drug free Procedure for preparation of mixture of Oseltamivir Phosphate and Ampicillin trihydrate: The concentration of Oseltamivir phosphate and Ampicillin trihydrate in the mixture was chosen on the basis of the dose of Ampicillin that is co-administered with Oseltamivir Both the drug 323

5 solutions equal to 15 ml was taken into the beaker and 15ml of albumin solution in boiling test separated by membrane.1ml of drug solution was taken from beaker for every 20mins and replaced with buffer immediately.1ml of solution was diluted to 10ml of buffer and absorbance was noted until a constant value by using UV spectrophotometer. From the protein binding studies of mixture of Oseltamivir phosphate and Ampicillin trihydrate the effect of Ampicillin trihydrate on the protein binding of Oseltamivir phosphate was determined. Table2, Fig 4 RESULTS Protein binding study of oseltamivir phosphate was carried out for 200 mins. The percentage of bound drug was found to be 80% and percentage of free drug was found to be 20%. Protein binding of oseltamivir was determined in the presence of Ampicillin trihydrate for 200 mins. The percentage of bound was found to be 68.6%and percentage of free drug was found to be 31.40%.Thus co-administration of Oseltamivir phosphate with Ampicillin trihydrate shows 57% increase in free drug concentration. CONCLUSION Invitro protein binding studies of Oseltamvir phosphate shows increased bioavailability of Oseltamivir phosphate in co-administration with Ampicillin trihydrate, thus protein binding of Oseltamivir phosphate was found to be altered in presence of Ampicillin trihydrate REFERENCES [1] G, Massarella J, Ward P. Clin Pharmacokinet (1999), 37: [2] Shargel, Leon. Applied Biopharmaceutics & Pharmacokinetics. New York: McGraw-Hill, Medical Pub. Division (2005). [3] Niklas Lindegardh et al., 2006, Antimicrobial Agents and Chemotherapy, September 2006, p , Vol. 50, No. 9 [4] P. Vijayaraghavan, Judith Justin. Experimental Biopharmaceutics and pharmacokinetics. New century book house,